Transcript THE MICROSCOPE In this exercise you will learn about the

The Microscope
In this exercise you will learn about the principles of optical microscopy and become familiar
with the use of the microscope. Microscopes are delicate and expensive instruments; they should
be handled with utmost care! Before you use the microscope, your instructor will explain its
proper use. Following are rules that will protect the microscope and insure that you can make
maximum use of it. Microscope safety rules are explained more thoroughly in the Biology
Department's Lab Safety Rules which you signed at the beginning of the semester.
Types of Microscopes
There are two different types of
microscopes: light and electron. Light
microscopes have glass lenses which
magnify objects, and light is necessary
to illuminate the objects being
examined. You will be using two
different kinds of light microscopes in
this lab, the compound microscope and
the dissecting microscope. Electron
microscopes use beams of electrons to
examine incredibly small objects (like
the components of an individual cell)
that have been specially prepared. The
different microscopes are explained in
more detail below.
The Compound Microscope
Compound microscopes are used to
examine objects in two dimensions.
Very small organisms or cross-sections
of organisms are placed on clear glass
slides; these objects are viewed as light
passes through them. The parts of the
compound microscope are reviewed
below.
The Dissecting Microscope
Dissecting microscopes are used to
observe material that is either too thick
or too large to be viewed with the
compound light microscope. While the
magnification and depth of field are
smaller in the dissect-
ing scope, the field of view is much
larger. As its name implies, the
dissecting scope is often used to dissect
plants, since it allows for manipulation
of material. Since most of the parts of
the dissecting microscope are the same
as the compound microscope, they will
not be reviewed here.
Electron Microscopy
The Biology Department has an electron
microscope suite, used for teaching
graduate-level students and for research.
In these microscopes a beam of
electrons (in place of light) and circular
magnets (in place of glass lenses) permit
the resolution of structures in much finer
detail than in an optical microscope. We
have two electron microscopes. The
first is a "traditional" transmission
electron microscope (TEM) in which
an electron beam passes through the
specimen. The second is the more
recently-developed scanning electron
microscope (SEM) in which a beam of
electrons scans the surface of an opaque
object and produces an image of that
surface. The images are viewed on a
cathode tube, or more critically by
exposing photographic film. Many of
the photographs of cell structure used in
your text were taken with an electron
microscope.
Rules for using the microscope- The Ten Commandments
1.
Always carry the microscope in a straight upright position with one hand around the arm
and the other hand under the base. The eyepieces are not attached and will fall out if the
microscope is carried at an angle or upside down.
2.
Check out the microscope to make sure all the lenses are clean and the mechanical parts are
in working order. Report any malfunction to the instructor so that it may be remedied.
3.
Keep the microscope clean. When anything is spilled or otherwise gets on the microscope,
clean it up immediately.
4.
When using the microscope start with the low power lens and work up to the desired
magnification. These microscopes are parfocal, which means that all powers should be in
focus when the turret is rotated.
5.
Never move the stage upwards with the coarse adjustment while viewing through the
eyepieces. Get the lens close to the slide while viewing from the side to make sure that they
never touch. Then move the stage downward with the coarse adjustment while viewing
through the lense. This will prevent the possibility of ramming the lens into the slide,
thereby ruining a slide you have just made and, quite possibly, damaging the lens.
6.
Moist, living or preserved materials must be observed through a coverslip. This protects the
lens as well as tends to make the object under view optically flat. Be sure to maintain a safe
distance between the coverslip and the objective lenses.
7.
Clean the lenses with lens paper only. DO NOT CLEAN THE LENSES WITH
HANDKERCHIEFS, FACIAL TISSUES, PAPER TOWELS, ETC.--they will scratch the
lenses. If your lenses are very dirty, obtain some lens cleaning solvent from the instructor.
8.
If you cannot obtain clear focus or good lighting, or if your microscope seems not to be
working properly, IMMEDIATELY CALL YOUR INSTRUCTOR. He/she can either assist
you or see that the microscope is repaired.
9.
Return your scope to the cabinet with light cord wrapped around its base and with the lowest
power objective lens in position.
10. Never leave any microscope slides on the microscope. Always remove them and return
them to their proper place before leaving the room.
THE COMPOUND MICROSCOPE
The Parts of a Compound Microscope
1. The microscope has two magnifying lenses: the eyepiece or ocular lens and the objective
lenses on a turret which revolves above the stage. The eyepiece lenses are usually 10X and
are moveable so that they can be adjusted to the distance between the pupils of each viewer.
The objective lenses (there are four: 4X, 10X, 40X and 100X) rotate on the nosepiece. By
changing the objectives the effective power of magnification is changed. The total
magnification observed is the product of the power of magnification of the eyepiece and the
objective. Only the 100X objective is used immersed in a drop of special oil (between the
lens and the slide; all others are designed to be used with air between the object and lens
surface. The 100X objective will not be used in this course. The power of magnification is
clearly indicated on each lens along with the numerical aperture of each lens. Depending
upon their design and quality, different objectives have different resolving distances. The
latter is the smallest distance between two points that allows both points to be viewed as
separate. This resolving distance is dependent upon the wavelength of light used as well as
the construction of the lens.
2. Microscopes contain elements designed to project parallel beams of light through the
specimen and into the objective. These include the projection lens which focuses light onto
the condenser lens. The condenser lens focuses light onto the object. To get the condenser
lens in focus, place a slide containing a wax pencil mark on the stage. Focus on it with the
lowest power objective lens and turn the iris diaphragm to the smallest opening. Then focus
the condenser up and down until the edges of the iris diaphragm come into sharp focus
without using the objective focusing adjustments. The condenser is now in focus.
3. The focusing knobs move the lens assembly up and down to bring the object in focus.
The coarse adjustment should only be used with the shortest, low power objective lens. The
fine adjustment (smaller knob) brings the object into critical focus. Notice that all objects
are projected upside down in the microscope field. It takes a little practice in using the
mechanical stage to move the slide where you want it.
Using the Compund Microscope
Use both hands to carry the microscope to your seat. Place the microscope on the table in front
of you and position yourself so that you are comfortably seated while looking through the
microscope.
If necessary, clean the lenses with lens paper only. Do not use anything else, like KimWipes or
your shirt to clean the lenses--this will damage the microscope.
3. Place a slide of the 'letter e' on the stage. If your microscope has a built in light, plug in the
scope and turn the light on. If not, bring a lamp to your table and position it so that the light
shines above the object being viewed.
4. Turn the nosepiece so that you are using the lowest power objective lens. You should always
use the lowest power objective when you begin viewing an object. While looking through the
ocular lenses with both eyes, begin to focus on the object by turning the focus adjustment on the
side of the microscope arm. If you see two images of the object or the reflection of your own
eye/eyelashes, you probably need to adjust the ocular lenses. These lenses can be moved
together or apart to better match the distance between your eyes.
5. Once the object is in focus, increase the magnification by rotating the nosepiece. Adjust the
focus by using the fine adjustment knob only. Make sure that the objective lens does not come
in contact with the slide.
6. Examine different parts of the object by moving it around the stage. Notice the direction that
the image moves when the object is moved from left to right. Change the light level and observe
differences in the way the image appears.
Additional Concepts
The field of view is the area visible when you look through the microscope. Knowing the
field of view will enable you to determine the size of the object you are observing. To
calculate the field of view, multiply the ojbective power by the ocular power (which is
always 10 in our microspcopes). There are also special rulers are used to determine the
field of view and measure objects under the microscope. Accurate measuring can be very
important when identifying plants or plant structures.
Drawing Objects To Scale: In drawing objects that you have seen with the microscope it
is important to describe how large they actually are. The actual magnification will depend
upon whether you have drawn "little" or "big" (you should draw "big"). The way to
estimate the actual size of the object is by knowing how wide the microscope's field of view
is. This can be estimated by using a scale that has been etched on a microscope slide.
Using this scale we have measured the width of each field for your microscopes:
4X x 10 = 4.6 mm, 4600 um
10X x10 = 1.8 mm, 1800 um
40X x 10 = 0.46 mm, 460 um
where μm = one micron, or one millionth of a meter
The Biology of Periphytons
You may have noticed that the ponds in the Miami area are frequently covered
with clumps of light-colored slime, what some might call “pond scum”. It might look pretty
disgusting to the average person, but to those of us who study the Everglades it is very special
stuff. We call it periphyton. It is a community of micro and macro-organisms that lives under the
water surface in the Everglades, or floats if it accumulates enough bubbles of oxygen. Periphyton
forms on the skeletons of flowering aquatic plants, particularly the bladderworts (genus
Utricularia). As a community periphyton consists of a variety of organisms that live in the
matrix of dead organic matter: bacteria, protozoans, green algae, diatoms, rotifers, insect larvae,
and much more. We have added several pages of illustrations of organisms that you can easily
find when you observe preparations with a microscope. This is also a good exercise for you to
learn how to use a microscope.
Periphyton is ecologically important in the Everglades because it is the source of
much of the carbon fixed in photosynthesis, and this is passed to other organisms, particularly
apple snails and small fish, in food webs. The snails and fish are eaten directly by birds, or often
by larger fish, that are then eaten by birds and alligators. So this is the stuff on which the
Everglades runs. A number of scientists at FIU are studying the effects of adding phosphorus (a
key ingredient in the water from the sugar cane farms to the north) on the function of the
wetlands ecosystems. We are finding that even modest additions of phosphorus cause the
periphyton to break apart. This may have unknown consequences for the function of this
ecosystem, and we are trying to figure this out.
Dead material
Alligators
Prawns
Crayfish
Periphyton
Gar
Rotifers
Copepods
Insect Larvae
Bass
Wading
Birds
Shellfish
Examining Periphyton Under the Microscope
In this laboratory exercise you will observe and identify organisms in the
periphyton community, taken from an Everglades pond, and supplied to the classroom. This is an
enjoyable process, because you may see amazingly bizarre living organisms swimming around in
the water, or non-moving green and photosynthetic algae. Try to match what you see to the
organisms illustrated here. Make a list of the organisms you have seen. If it is unusually
interesting, share the view with your table partners.
Examining Microorganisms
Take a piece of the periphyton mat without squeezing it and place it in a petri dish. (Sometimes
there are flies in the periphyton that will bite if you squeeze the periphyton too hard.) Place the
dish on a dissecting microscope and examine the periphyton for macro-organisms. Use the
following diagrams to help you identify what you are observing. If possible, try to isolate a
few of the more interesting organisms by using tweezers.
Clam or Mussel
Leech
Adult Beetle
Rotifer
Mayfly larva
Copepod
Water Mite
Hemiptera
(Water Bug)
Midge larva
Gammarus (Scud)
Stonefly Larva
Snail
Examining Microorganisms
In order to see micro-organisms present in the periphyton, it needs to be homogenized
(ground up or pureed), diluted with water, and examined under a compound light
microscope. This has already been completed for you. Place one drop of homogenized
periphyton on a microscope slide. Gently add a cover slip, by placing one edge against the
slide and allowing it to fall over the tissue. This helps force out the air bubbles that tend to
be trapped under the coverslip. You can remove excess water by twisting an end of a
kleenex (or kimwipe) and placing it on the edge of the coverslip. It will absorb the excess
water. Then place the slide on the microscope stage and begin your observations under low
power (10X). Look for a variety of micro-organisms, as illustrated in the following pages,
in the periphyton. You can boost the power by turning the nosepiece to a higher power
objective (watch your instructor demonstrate this). You can estimate the size of the
organism by comparing its length to the diameter of the field at any given magnification.
If you see absolutely nothing, then try preparing another slide of periphyton, then look
again.
PROTOZOANS
Euglena
Volvox
Peridinium
GREEN ALGAE
Chlamydomonas
Bulbochaete
Mougeotia
Spirogyra
Oedogonium
Ulothrix
DIATOMS
Gomphonema
Frustulia
Navicula
Fragilaria
Nitzchia
CYANOBACTERIA
DESMIDS
Nostoc
Oscillatoria
Desmidium
Experimenting on Periphytons
Using the techniques you have learned in the lab you could ask some interesting questions
about periphyton, and collect observations consistent or inconsistent with the hypotheses
stemming from these questions. Here are some sample questions.
1. Light levels at the top should be much higher than at the bottom of a floating mat of
periphyton. Organisms adapted to different light intensities should be found at different
levels in the mat. You could simply sample different levels of the mat and count the
organisms you have observed.
2. Organisms adapted to specific light levels may move vertically up and down in the mat
during the day. You could count organisms at different levels and at different times of the
day.
3. Conditions, as water temperature and sunlight, change during the year. Periphyton
organisms should change in abundance at different times of the year. Again, you could
count organisms in mats at different times of the year.
Fossils
Fossils are remnants of once living organisms. Fossils are direct evidence for organisms that
lived in the distant past. Fossils were produced under optimal conditions and only by
organisms with hard body parts that allowed such formation. These were places on the
earth where dead organisms were covered by fine sediments, oxygen was excluded from
oxidizing the structures, and the organisms were thus preserved. Fossils are only found in
sedimentary rocks. Fossils may be impressions or compressions of once-living organisms.
Hard structures, as shells or skeletons may be fossilized directly. Other fossils are formed
with minerals gradually replace the once-living tissues in a process termed petrifaction, like
petrified wood. Fossils are the best evidence available on the organisms that were present
in the distant past. In this display, observe the fossils on display. For each fossil determine
its age (approximately in millions of years before present) by matching the eras during
which the organisms lived with the time scale on the poster. Determine the process by
which each fossil was formed. Finally, determine the phylum and kingdom to which each
fossil belonged.
AMMONITES
These animal fossils were formed because of the hard calcareous shell secreted by
the organisms. These are members of the Cephalopoda. What living organisms do
you think these organisms were related to? These organisms lived during the
Mesozoic era.
TRILOBITES
These are the most ancient of the organisms displayed among these fossils, dating
back to the Cambrian era. These fossils were formed because of a hard
exoskeleton, so durable that the shape of the fossil gives a good idea of the three
dimensional shape of the organism. The exoskeleton consists of segments, with
many small appendages. What living organisms do you think these organisms
were related to?
LEPIDODENDRON
This is a plant fossil, common in the coal deposits of Pennsylvania and West Virginia, dating
back to the Carboniferous. What you are looking at is the surface of the trunk of a small tree,
and the diamond shaped structures are the scars where leaves were once attached.
More Fossils
LEPIDOSTROBUS
This is also a plant fossil, common in the coal deposits of Pennsylvania and West
Virginia, dating back to the Carboniferous. You are looking at the surface of a
cone-like structure of a plant that did not produce seeds. This name is the example
of a form genus. It was given its name as a structure. Later on other fossils were
found that connected it to the stem, Lepidodendron. Later on another form genus
was established for the roots. Yet these are all fossils of a single plant species.
These fossils are of plants that were related to the living genus Lycopodium. This
genus is often given the name of a “living fossil” because of its ancient origins.
CALAMITES
This is a plant fossil, common in the coal deposits of Pennsylvania and West Virginia,
dating back to the Carboniferous. You are looking at the surface of a stem whose
branches all originate at a single node, all the way around the stem. On the small
branches, leaves also appear at the nodes. These plants produced spores, on cone-like
structures, and did not produce seeds. These fossils are of plants that were related to the
living genus Equisetum, or the scouring rush.
PETRIFIED WOOD
These are various samples of wood, deposited in sandstones in deserts of the southwest.
These are the youngest of fossils on display, produced during the Tertiary. Wood
anatomists can trace the arrangements of cells in these fossils to genera of conifers
living today. Thus the names on the fossils correspond to plants you may be familiar
with, such as Taxodium (cypress). What are desert localities today once supported
coniferous forests back then. If you look carefully at parts of the wood you can see the
annual growth rings of the wood. The thickness of these rings can also analyzed to
estimate rates of growth and the types of climates in which these trees lived.
Survey of Bacteria
Cellular organisms have evolved along two lines. Species with cells lacking membraneBound organelles are prokaryotes. Those with membrane-bound organelles are eukaryotes and
include plants, animals, fungi, and protists. About 5000 species of prokaryotes have been
described, and many more await identification.
Prokaryotes were long thought to be a unified group commonly called bacteria. However,
genetic analysis as recently as 1996 of the DNA of prokaryotes has revealed two groups with
surprisingly different DNA sequences, both of which are strikingly different from the DNA
sequences of eukaryotes. This has led to recognition of three domains of organisms.
Domain Archaea include kingdom Archaebacteria, all species of which are prokaryotes.
Archaebacteria often inhabit but are not restricted to extreme and stressful environments on earth.
Domain Bacteria includes kingdom Bacteria, all species of which are prokaryotes and are the
most abundant organisms on earth. Domaine Eukarya includes kingdoms Protista, Fungi,
Plantae, and Animalia. These kingdoms are all eukaryotes. This classification of living
organisms into three domains and six kingdoms is widely accepted, but much phylogenetic
information remains to be revealed. Classification is an exciting and ongoing process.
Kingdom Archaebacteria
Kingdom Bacteria
Archaebacteria of domain Archaea may
be the oldest form or life an earth, an domains
Bacteria and Eukarya probably diverged
from Archaebacteria independently.
Archaebacteria are diverse prokaryotes
that share ribosomal RNA sequences as well
as several important biochemical
characteristics that are quite distinctive from
those of all other kinds of organisms.
Archaebacteria have distinctive membranes,
unusual cell walls, and unique metabolic
cofactors.
Today’s Archaebacteria are probably
survivors on ancient lines that have persistent
in habitats similar to habitats found throughout
the world when bacteria first evolved. These
environments are often extremely acidic, hot, or
salty. Thus, many Archaebacteria are called
extremophiles. Many Archaebacteria can live in
an anaerobic atmosphere rich in carbon dioxide
and hydrogen as well as the more benign
environments.
Bacteria of the kingdom Bacteria are distributed
more widely than any other group of organisms.
Individual bacteria cells are microscopic (1 m
or less in diameter); a single gram of soil may
contain over a billion bacteria. Bacteria have
cell walls, which give them three characteristic
shapes. (See figure on next page for example of
the different bacteria shapes.)
•
•
•
Bacillus (rod-shaped)
Coccus (sphirical)
Spirillum (spiral)
Most bacteria are heterotrophic, meaning that
they derive their energy from organic molecules
made by other organisms. Heterotrophic
bacteria are decomposers because they feed on
dead organic matter and release nutrients locked
in dead tissue. Bacteria that derive their energy
from photosynthesis or the oxidation of
inorganic molecules are called autotrophic.
However, photosynthesis in bacteria is often
different from that in eukaryotes, because sulfur
rather than oxygen is sometimes produced as a
by-product.
The prokaryotes: Bacteria
Here are some examples of the 3 shapes
of bacteria.
•Bacillus = rod-shaped
•Streptococcus = coccus (round)
•Spirillum = spiral
Although bacteria can reproduce
sexually through a process called
conjugation, their mode of reproduction
is almost always asexual, by simple
binary fission.
Nitrogen fixing bacteria- Certain
bacteria and cyanobacteria transform
atmospheric nitrogen (N2) into other
nitrogenous compounds that can be used as
nutrients by plants. This process in called
nitrogen fixation. All organisms need nitrogen
as a component of their nucleic acids, proteins,
and amino acids. However, only certain
bacteria and cyanobacteria have the capability
to break the bonds between atmospheric
nitrogen and convert it to ammonia (NH3)
which can be used by plants. These bacteria
form nodules on the plant roots where they
convert the nitrogen in exchange for sugar and
other nutrients provided by the plant. Such an
association, where both organisms benefit, is
known as symbiosis.
Root nodules containing nitrogen-fixing bacteria
Some Important Prokaryotes
Cyanobacteria
A member of the kingdom Bacteria, these photosynthetic prokaryotes, formerly called bluegreen algae, were the first organisms to fix carbon dioxide and produce oxygen in
photosynthesis. They transformed the early atmosphere from reducing to oxygen-rich.
They continue to be important today in many ecosystems. Cyanobacteria, both filamentous
and single to multiple cell organisms, are an important portion of the periphyton
communities in the Everglades.
Cyanobacteria
Lactobacillus
These bacteria modify milk in the production of yogurt. Some people also take them as a
dietary supplement to improve their digestion, particularly after having received a dose of
antibiotics. They can easily be observed in yogurt that contains live cultures of this
bacterium, as you will discover in one of your exercises.
Fern Spore Germination
Ferns are a part of the plant kingdom, but do not produce flowers, fruits, or seeds like
many other plants. Instead, ferns produce spores on the underside of leaves. These
spores are dispersed by wind and will germinate if they land on moist soil.
Eventually each spore develops into a flat, heart-shaped structure called a thallus.
Once fertilization occurs, a new fern starts to grow from the thallus. Over time, this
new fern becomes much larger than the thallus and takes on the fern form that we are
familiar with. In this procedure, you will be sowing spores of the fern Ceratopteris,
a very rapidly growing fern. In two or three days, the spores will germinate and after
10-12 days, thalli should be apparent. Throughout the semester, you will observe the
development of the fern spores and record your observations. The following
procedures are based on those provided by the C-Fern company and are protected by
copyright laws.
Procedure:
1. Use small Styrofoam containers, such as those used for soup take out. There should be
one container per table, with a total of 6 per lab. Fill it with sterilized potting soil to
a depth of about 2.5 cm (or 1 inch).
2. Gently moisten the soil with the small spray bottle.
3. Using a cotton swab (or q-tip), place the cotton tip into the vial of fern spores.
4. Place the cotton tip above the soil, tap the shaft of the q-tip as you move the cotton tip
around on the soil of the dish.
5. Puncture two small holes in the lid of the dish, and label it using the label tape and
marker. Indicate your lab group, the lab section and the date.
6. Place the plastic dish on the shelf indicated by your instructor, under the fluorescent
lights.
7. Observe the changes in the development of the spores each week during the term. Note
the numbers of cells and their arrangement. Document with simple sketches.